Individual variation and longitudinal pattern of genome-wide DNA methylation from birth to the first two years of life

Prenatal development and early childhood are critical periods for establishing the tissue-specific epigenome, and may have a profound impact on health and disease in later life. However, epigenomic profiles at birth and in early childhood remain largely unexplored. The focus of this report is to examine the individual variation and longitudinal pattern of genome-wide DNA methylation levels from birth through the first two years of life in 105 Black children (59 males and 46 females) enrolled at the Boston Medical Center. We performed epigenomic mapping of cord blood at birth and venous blood samples from the same set of children within the first two years of life using Illumina Infinium Humanmethylation27 BeadChip. We observed a wide range of inter-individual variations in genome-wide methylation at each time point including lower levels at CpG islands, TSS200, 5′UTR and 1st Exon locations, but significantly higher levels in CpG shores, shelves, TSS1500, gene body and 3′UTR. We identified CpG sites with significant intra-individual longitudinal changes in the first two years of life throughout the genome. Specifically, we identified 159 CpG sites in males and 149 CpG sites in females with significant longitudinal changes defined by both statistical significance and magnitude of changes. These significant CpG sites appeared to be located within genes with important biological functions including immunity and inflammation. Further studies are needed to replicate our findings, including analysis by specific cell types, and link those individual variations and longitudinal changes with specific health outcomes in early childhood and later life.     

人类基因组DNA甲基化数据分析的研究现状

作为人类基因组最为典型的表观遗传现象,DNA甲基化在多种关键生理活动中扮演重要角色.系统分析基因组尺度的DNA甲基化概况意义重大.从Cp G岛等基本定义出发,阐述了高通量DNA甲基化的检测技术以及针对芯片技术与下一代测序技术的低水平数据处理方法;重点对比了基于机器学习理论对Cp G位点及Cp G岛甲基化水平的预测算法,以及所利用的特征对预测效果的影响与发展趋势;并对DNA差异甲基化在组织特异性、癌症等多种疾病中的计算分析进行了全面的综述.     

Whole blood DNA aberrant methylation in pancreatic adenocarcinoma shows association with the course of the disease: a pilot study

Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient's blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors.     

Heritability of DNA methylation in threespine stickleback (Gasterosteus aculeatus)

Epigenetic mechanisms underlying phenotypic change are hypothesized to contribute to population persistence and adaptation in the face of environmental change. To date, few studies have explored the heritability of intergenerationally stable methylation levels in natural populations, and little is known about the relative contribution of cis- and trans-regulatory changes to methylation variation. Here, we explore the heritability of DNA methylation, and conduct methylation quantitative trait loci (meQTLs) analysis to investigate the genetic architecture underlying methylation variation between marine and freshwater ecotypes of threespine stickleback (Gasterosteus aculeatus). We quantitatively measured genome-wide DNA methylation in fin tissue using reduced representation bisulfite sequencing of F1 and F2 crosses, and their marine and freshwater source populations. We identified cytosines (CpG sites) that exhibited stable methylation levels across generations. We found that additive genetic variance explained an average of 24–35% of the methylation variance, with a number of CpG sites possibly autonomous from genetic control. We also detected both cis- and trans-meQTLs, with only trans-meQTLs overlapping with previously identified genomic regions of high differentiation between marine and freshwater ecotypes. Finally, we identified the genetic architecture underlying two key CpG sites that were differentially methylated between ecotypes. These findings demonstrate a potential role for DNA methylation in facilitating adaptation to divergent environments and improve our understanding of the heritable basis of population epigenomic variation.     

The Dynamics of DNA Methylation Covariation Patterns in Carcinogenesis

Recently it has been observed that cancer tissue is characterised by an increased variability in DNA methylation patterns. However, how the correlative patterns in genome-wide DNA methylation change during the carcinogenic progress has not yet been explored. Here we study genome-wide inter-CpG correlations in DNA methylation, in addition to single site variability, during cervical carcinogenesis. We demonstrate how the study of changes in DNA methylation covariation patterns across normal, intra-epithelial neoplasia and invasive cancer allows the identification of CpG sites that indicate the risk of neoplastic transformation in stages prior to neoplasia. Importantly, we show that the covariation in DNA methylation at these risk CpG loci is maximal immediately prior to the onset of cancer, supporting the view that high epigenetic diversity in normal cells increases the risk of cancer. Consistent with this, we observe that invasive cancers exhibit increased covariation in DNA methylation at the risk CpG sites relative to normal tissue, but lower levels relative to pre-cancerous lesions. We further show that the identified risk CpG sites undergo preferential DNA methylation changes in relation to human papilloma virus infection and age. Results are validated in independent data including prospectively collected samples prior to neoplastic transformation. Our data are consistent with a phase transition model of carcinogenesis, in which epigenetic diversity is maximal prior to the onset of cancer. The model and algorithm proposed here may allow, in future, network biomarkers predicting the risk of neoplastic transformation to be identified.     

Sex-specific effects of early life cadmium exposure on DNA methylation and implications for birth weight

Dietary cadmium exposure was recently found to alter DNA methylation in adults, but data on effects early in life are lacking. Our objective was to evaluate associations between prenatal cadmium exposure, DNA methylation and birth weight. In total 127 mother-child pairs from rural Bangladesh were studied. For comparison, we included 56 children at 4.5 y. Cadmium concentrations in mothers’ blood (gestational week 14) and children’s urine were measured by ICPMS. Global DNA methylation was analyzed by Infinium HumanMethylation450K BeadChip in cord blood and children’s blood. Maternal cadmium exposure was associated with cord blood DNA methylation (p-value < 10^–16). The association was markedly sex-specific. In boys, 96% of the top 500 CpG sites showed positive correlations (r_S-values > 0.50), whereas most associations in girls were inverse; only 29% were positive (r_S > 0.45). In girls we found overrepresentation of methylation changes in genes associated with organ development, morphology and mineralization of bone, whereas changes in boys were found in cell death-related genes. Several individual CpG sites that were positively associated with cadmium were inversely correlated with birth weight, although none statistically significant after correction for multiple comparisons. The associations were, however, fairly robust in multivariable-adjusted linear regression models. We identified CpG sites that were significantly associated with cadmium exposure in both newborns and 4.5-y-old children. In conclusion, cadmium exposure in early life appears to alter DNA methylation differently in girls and boys. This is consistent with previous findings of sex-specific cadmium toxicity. Cadmium-related changes in methylation were also related to lower birth weight.     

Rapid and specific hypomethylation of enhancers in endothelial cells during adaptation to cell culturing

Epigenetics, including DNA methylation, is one way for a cell to respond to the surrounding environment. Traditionally, DNA methylation has been perceived as a quite stable modification; however, lately, there have been reports of a more dynamic CpG methylation that can be affected by, for example, long-term culturing. We recently reported that methylation in the enhancer of the gene encoding the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) was rapidly erased during cell culturing. In the present study we used sub-culturing of human umbilical vein endothelial cells (HUVECs) as a model of environmental challenge to examine how fast genome-wide methylation changes can arise. To assess genome-wide DNA methylation, the Infinium HumanMethylation450 BeadChip was used on primary, passage 0, and passage 4 HUVECs. Almost 2% of the analyzed sites changed methylation status to passage 4, predominantly displaying hypomethylation. Sites annotated as enhancers were overrepresented among the differentially methylated sites (DMSs). We further showed that half of the corresponding genes concomitantly altered their expression, most of them increasing in expression. Interestingly, the stroke-related gene HDAC9 increased its expression several hundredfold. This study reveals a rapid hypomethylation of CpG sites in enhancer elements during the early stages of cell culturing. As many methods for methylation analysis are biased toward CpG rich promoter regions, we suggest that such methods may not always be appropriate for the study of methylation dynamics. In addition, we found that significant changes in expression arose in genes with enhancer DMSs. HDAC9 displayed the most prominent increase in expression, indicating, for the first time, that dynamic enhancer methylation may be central in regulating this important stroke-associated gene.     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

Food allergy is mediated by a combination of genetic and environmental risk factors, potentially mediated by epigenetic mechanisms. CD4+ T-cells are key drivers of the allergic response, and may therefore harbor epigenetic variation in association with the disease phenotype. Here we retrospectively examined genome-wide DNA methylation profiles (~450?000 CpGs) from CD4+ T-cells on a birth cohort of 12 children with IgE-mediated food allergy diagnosed at 12-months, and 12 non-allergic controls. DNA samples were available at two time points, birth and 12-months. Case:control comparisons of CD4+ methylation profiles identified 179 differentially methylated probes (DMP) at 12-months and 136 DMP at birth (FDR-adjusted P value < 0.05, delta β > 0.1). Approximately 30% of DMPs were coincident with previously annotated SNPs. A total of 96 allergy-associated non-SNP DMPs were present at birth when individuals were initially disease-free, potentially implicating these loci in the causal pathway. Pathway analysis of differentially methylated genes identified several MAP kinase signaling molecules. Mass spectrometry was used to validate 15 CpG sites at 3 candidate genes. Combined analysis of differential methylation with gene expression profiles revealed gene expression differences at some but not all allergy associated differentially methylated genes. Thus, dysregulation of DNA methylation at MAPK signaling-associated genes during early CD4+ T-cell development may contribute to suboptimal T-lymphocyte responses in early childhood associated with the development of food allergy.     

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

Food allergy is mediated by a combination of genetic and environmental risk factors, potentially mediated by epigenetic mechanisms. CD4+ T-cells are key drivers of the allergic response, and may therefore harbor epigenetic variation in association with the disease phenotype. Here we retrospectively examined genome-wide DNA methylation profiles (~450 000 CpGs) from CD4+ T-cells on a birth cohort of 12 children with IgE-mediated food allergy diagnosed at 12-months, and 12 non-allergic controls. DNA samples were available at two time points, birth and 12-months. Case:control comparisons of CD4+ methylation profiles identified 179 differentially methylated probes (DMP) at 12-months and 136 DMP at birth (FDR-adjusted P value < 0.05, delta β > 0.1). Approximately 30% of DMPs were coincident with previously annotated SNPs. A total of 96 allergy-associated non-SNP DMPs were present at birth when individuals were initially disease-free, potentially implicating these loci in the causal pathway. Pathway analysis of differentially methylated genes identified several MAP kinase signaling molecules. Mass spectrometry was used to validate 15 CpG sites at 3 candidate genes. Combined analysis of differential methylation with gene expression profiles revealed gene expression differences at some but not all allergy associated differentially methylated genes. Thus, dysregulation of DNA methylation at MAPK signaling-associated genes during early CD4+ T-cell development may contribute to suboptimal T-lymphocyte responses in early childhood associated with the development of food allergy.     

Methylation Levels of SLC23A2 and NCOR2 Genes Correlate with Spinal Muscular Atrophy Severity

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder subdivided into four different types. Whole genome methylation analysis revealed 40 CpG sites associated with genes that are significantly differentially methylated between SMA patients and healthy individuals of the same age. To investigate the contribution of methylation changes to SMA severity, we compared the methylation level of found CpG sites, designed as “targets”, as well as the nearest CpG sites in regulatory regions of ARHGAP22, CDK2AP1, CHML, NCOR2, SLC23A2 and RPL9 in three groups of SMA patients. Of notable interest, compared to type I SMA male patients, the methylation level of a target CpG site and one nearby CpG site belonging to the 5’UTR of SLC23A2 were significantly hypomethylated 19–22% in type III-IV patients. In contrast to type I SMA male patients, type III-IV patients demonstrated a 16% decrease in the methylation levels of a target CpG site, belonging to the 5’UTR of NCOR2. To conclude, this study validates the data of our previous study and confirms significant methylation changes in the SLC23A2 and NCOR2 regulatory regions correlates with SMA severity.